EFFECTS OF HUMAN LEUKOCYTE CONDITIONED MEDIUM ON MOUSE HAEMOPOIETIC PROGENITOR CELLS

Medium conditioned by human peripheral blood leukocytes (HLCM) was studied for its in vitro effects on haemopoietic progenitor cells (CFU-s and CFU-c) present in mouse bone marrow. HLCM has poor colony stimulating activity in semi-solid cultures of mouse bone marrow cells. but invariably increases the number of colonies obtained in the presence of plateau levels of semi-purified colony stimulating factor (CSF). In liquid cultures, HLCM appears to contain a potent initiator of DNA synthesis in CFU-s. an activity which coincides with an increased CFU-s maintenance and causes a three- to four-fold increase in CFU-c number. It is apparent from this study that HLCM, in addition to stimulating colony formation in cultures of human bone marrow cells, has a profound in vitro effect on primitive haemopoietic progenitor cells of the mouse, which cannot be attributed to CSF.     

THE PRESENCE OF A CSF ENHANCING ACTIVITY IN THE SERUM OF ENDOTOXIN-TREATED MICE

Serum taken from mice a few hours after injection of endotoxin is a potent source of a stimulator of in vitro myelopoiesis. By means of dose-response studies, the biological activity of this material was compared to that of a colony stimulating factor (CSF) from pregnant mouse uteri. Postendotoxin serum appears to contain two different activities: a stimulating activity which may be identical to CSF and an activity which augments the action of CSF. The separate nature of the two activities is demonstrated by differences in the rate at which they are diluted out and by differences in the time at which they are maximally present after endotoxin administration. It is therefore concluded that the colony-stimulating properties of postendotoxin serum are not due solely to CSF present in the serum.     

The effects of acylation stimulating protein supplementation <Emphasis Type="Italic">VS</Emphasis> antibody neutralization on energy expenditure in wildtype mice

Background  

Acylation stimulating protein (ASP) is an adipogenic hormone that stimulates triglyceride (TG) synthesis and glucose transport in adipocytes. Previous studies have shown that ASP-deficient C3 knockout mice are hyperphagic yet lean, as they display increased oxygen consumption and fatty acid oxidation compared to wildtype mice. In the present study, antibodies against ASP (Anti-ASP) and human recombinant ASP (rASP) were tested in vitro and in vivo. Continuous administration for 4 weeks via osmotic mini-pump of Anti-ASP or rASP was evaluated in wildtype mice on a high-fat diet (HFD) to examine their effects on body weight, food intake and energy expenditure.     

Mechanisms towards compensation of long-term haemopoietic injury in mice after 5 Gy irradiation:In vivo andin vitro enhancement of superoxide anion production by granulocytes

This paper analyzes the long-term (6 and 12 months) function of mouse granulocytes after total body irradiation with a single dose (5 Gy) of X-rays. Superoxide anion production has been investigated in granulocytes from peripheral blood, and also in those harvested from long term bone marrow cultures, with the aim of correlating the environmental damage induced by radiation with the functional properties of granulocytes. Anin vivo andin vitro enhancement of superoxide anion production and protein levels in granulocytes from irradiated mice is described. The presence of some colony stimulating factor in the supernatant of cultures from irradiated mice could play an important role in the priming of granulocytes.     

METALLIC COPPER-INDUCED GRANULOCYTE EXUDATION IN THE STUDY OF GRANULOCYTOPOIESIS

C3H/HeHa mice implanted with rods of metallic copper (CR) or glass (GR) exudate neutrophil granulocytes and mononuclear cells into the site of rod implant (peritoneal cavity). Exudation in CR mice is substantially greater than in GR animals. In CR mice there is an impressive stimulation of myelopoiesis measured in the femoral marrow subsequent to the initial accumulation in the peritoneal cavity. Serum levels of colony stimulating activity (CSA), an in vitro myeloproliferative stimulating activity, are elevated in such animals, as are femoral marrow agar-colony forming cells (CFC). The procedure is useful to the study of myelopoiesis and myelopoietic regulatory mechanisms.     

Tissue sources of bone marrow colony stimulating factor

Possible tissue sources in C57BL mice of the serum factor stimulating colony formation in vitro by mouse bone marrow cells have been investigated. A reproducible technique employing batch chromatography on calcium phosphate gel was developed for the extraction and assay of material with colony stimulating activity from mouse tissues. Sixteen hematopoietic and non-hematopoietic tissues from C57BL mice were found to vary widely in their content of extractable activity. Characterisation of the colony stimulating factors (CSF's) from these tissues by assay of stepped concentrations of eluate showed that CSF's from most tissues were similar in chromatographic behavior, but all differed significantly from those of serum in being both more disperse and more firmly bound to calcium phosphate gel. Male submaxillary salivary gland gave the richest yield of CSF. CSF from this source displayed a greater dispersity on and affinity to calcium phosphate, a lower electrophoretic mobility and a smaller average sedimentation coefficient than that from any other source investigated. Colony morphology appeared to be identical for all tissue sources investigated.     

Trypanosoma cruzi: In vivo and in vitro correlation between T-cell activation and susceptibility in inbred strains of mice

The in vivo susceptibility of several inbred strains of mice to the Y and CL strains of Trypanosoma cruzi was compared to the in vitro ability of spleen cells from infected mice to generate factor(s) able to activate macrophages to a trypanocidal state. Spleen cells from resistant immune mice generate higher levels of the factor(s) and do so at earlier times during infection than those of susceptible mice. The spleen cells capable of generating the in vitro factor(s) are also capable of conferring resistance upon passive transfer. Removal of immunoglobulin-bearing cells from the immune spleen cell population did not affect either transfer of protection in vivo or generation of the factor(s) in vitro. The cellular basis underlying the differences between susceptible and resistant mouse strains has not yet been determined.     

Activation of Murine Macrophages by Polysaccharide Fractions from Marine Algae (Porphyra yezoensis)

Two discrete immunomodulating fractions were obtained from marine algae (Porphyra yezoensis): one was the Porphyra water-soluble fraction (PWSF) which was extracted with hot water from the whole body of algae, and the other was the Porphyra acid-soluble fraction (PASF) which was extracted with acid from the residue. The major constituent in both PWSF and PASF was a polysaccharide, the total sugar concentration in PWSF (56.4%) being lower than that in PASF (82.2%). The high contents of 3,6-anhydrogalactose and sulfate indicated the porphyran structure in PWSF and PASF. The results of an in vitro culture assay with proteose peptone-induced macrophages from mice revealed that PWSF and PASF both enhanced glucose consumption, as well as the production of nitrite and tumor necrosis factor (TNF), but that these were increased more by PWSF than by PASF. PWSF augmented IL-1 secretion from these macrophages, while PASF did not. On the other hand, the carbon clearance activity of phagocytes from mice injected intraperitoneally with PASF was higher than that from PWSF-injected mice. The injection of PASF into mice also enhanced the carbon clearance activity in a dose-dependent manner. These results suggest that the two individual fractions possessed the ability to activate macrophages in vitro and in vivo in different ways.     

Stimulating Factors and Cell Recruitment In Murine Bone Marrow Stem Cells and Emt6 Tumours

The role of a stimulating factor in cell recruitment and the kinetics of its secretion were investigated by in vivo and in vitro techniques. the association of these two methods made it possible to demonstrate that a non-cycling population liberates a factor which in turn stimulates quiescent bone marrow stem cells into DNA synthesis. Moreover, it seems that undamaged cells are capable of secreting this factor. A stimulating factor responsible for cell recruitment was also demonstrated in an experimental EMT6 tumour and the kinetics of its secretion reported.     

Oocytes Preserve the Ability of Mouse Cumulus Cells in Culture to Synthesize Hyaluronic Acid and Dermatan Sulfate

A soluble factor(s) produced by fully grown oocytes is essential, together with follicle stimulating hormone (FSH), to stimulate in vitro hyaluronic acid (HA) synthesis by mouse cumulus cells (CCs). The stability of the response to this stimulus by CCs in culture was investigated. The data showed that preculture for 8 hr in basal medium reduced to approximately 30% the ability of CCs to synthesize HA in response to FSH or dibutyryl cyclic AMP (Bt₂cAMP) and soluble oocyte factor(s). However, if CCs were precultured for the same period of time as intact cumulus cell-oocyte complexes, or in the presence of fully grown oocytes, or in medium conditioned by fully grown oocytes, their ability to synthesize HA was 75-95% preserved. In vitro stimulation of dermatan sulfate (DS) synthesis by CCs does not require oocyte factors and is induced by FSH or Bt₂cAMP treatment alone. However, the preservation of such activity, like that of HA synthesis, depended on the presence of a soluble oocyte factor(s) during preculture. The presence of isolated oocytes or of oocyte-conditioned medium also prevented the spreading of CCs in culture. However, inhibiting CC spreading by culture on agar-coated plates or in serum-free medium did not preserve their HA or DS synthetic activity, thus suggesting that the two oocyte actions on CCs are independent. Growing oocytes were unable both to induce HA synthesis in freshly isolated CCs stimulated with FSH and to preserve the ability to synthesize HA and DS in 8-hr precultured CCs. The results suggest that the stability of the differentiated state of mouse CCs in vitro depends upon continued exposure to a soluble factor(s) produced by fully grown oocytes.     

Colony stimulating activity of serum from germfree normal and leukemic mice

Serum from germfree Swiss/HaM mice exhibited a reduced capacity to stimulate granulocytic and mononuclear cell colony formation by DBA/1 bone marrow cells in vitro when compared with serum from conventional Swiss/HaM mice. Sera from germfree preleukemic and leukemic AKR mice exhibited strong colony stimulating activity, indicating that the increased colony stimulating activity previously observed in the serum of conventional leukemic mice is not the consequence of bacterial or fungal infections supervening in leukemic animals with deficient immune responses.     

The eIF2A knockout mouse

Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the early 1970s as a factor capable of stimulating initiator methionyl-tRNAi (Met-tRNA^Met_i) binding to 40S ribosomal subunits in vitro. However, in contrast to the eIF2, which stimulates Met-tRNA^Met_i binding to 40S ribosomal subunits in a GTP-dependent manner, eIF2A didn't reveal any GTP-dependence, but instead was found to direct binding of the Met-tRNA^Met_i to 40S ribosomal subunits in a codon-dependent manner. eIF2A appears to be highly conserved across eukaryotic species, suggesting conservation of function in evolution. The yeast Saccharomyces cerevisae eIF2A null mutant revealed no apparent phenotype, however, it was found that in yeast eIF2A functions as a suppressor of internal ribosome entry site (IRES)-mediated translation. It was thus suggested that eIF2A my act by impinging on the expression of specific mRNAs. Subsequent studies in mammalian cell systems implicated eIF2A in non-canonical (non-AUG-dependent) translation initiation events involving near cognate UUG and CUG codons. Yet, the role of eIF2A in cellular functions remains largely enigmatic. As a first step toward characterization of the eIF2A function in mammalian systems in vivo, we have obtained homozygous eIF2A-total knockout (KO) mice, in which a gene trap cassette was inserted between eIF2A exons 1 and 2 disrupting expression of all exons downstream of the insertion. The KO mice strain is viable and to date displays no apparent phenotype. We believe that the eIF2A KO mice strain will serve as a valuable tool for researchers studying non-canonical initiation of translation in vivo.     

THYMIDINE SUICIDE IN VIVO AND IN VITRO OF SPLEEN COLONY FORMING AND AGAR COLONY FORMING CELLS OF MOUSE BONE MARROW

The ‘thymidine suicide’technique for indicating differences in the proliferation rate of early haemopoietic progenitor cells (spleen colony forming and agar colony forming cells) in C57BL mice has been evaluated. Special care was taken to use the same bone marrow cell suspension for the two progenitor cell assays. Both the in vivo and the in vitro techniques were employed. Following ³H-TdR in vivo, about 20% of both types of progenitor cell are killed in normal mice; however, after incubation in vitro with ³H-TdR, 35% of agar colony forming cells but only 4% of spleen colony forming cells are killed. Reasons for the difference between the in vivo and the in vitro results are discussed. With bone marrow from continuously irradiated animals, the thymidine suicide for both agar colony forming and spleen colony forming cells is in the range 42–50%, and there is no difference between in vivo and in vitro suicide. The in vivo results support the conclusion, based on the effect of proliferation dependent cytotoxic agents, that in C57BL mice agar colony forming and spleen colony forming cells are proliferating at the same rate in normal animals, and are speeded up to the same extent by continuous γ-irradiation. It is considered that in normal C57BL mice the in vitro method does not give a correct estimate of the proliferation rate of these progenitor cells. It would seem that the similarity in the proliferation rate of agar colony forming and spleen colony forming cells in C57BL mice is not true for other strains of mice: indeed using normal CBA and in vivo suicide, we have shown a significantly greater thymidine suicide for agar colony forming cells compared to spleen colony forming cells.     

Effects of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) on Biomaterial-Associated Staphylococcal Infection in Mice

Staphylococcal infections are a major complication in the usage of biomaterials. Different modifications of polymers have been made to reduce the incidence of such infections. We studied the effects of modifying heparinized polyethylene (H-PE) with mouse recombinant granulocyte-macrophage stimulating factor (rGM-CSF). The elimination of staphylococci (Staphylococcus aureus, S. epidermidis) from the peritoneum of mice implanted with rGM-CSF-coated H-PE was slightly more effective than the elimination of the bacteria from the peritoneum of animals implanted with uncoated H-PE. Most interestingly, the number of staphylococci present in the biofilms covering rGM-CSF-coated implants were significantly lower than the number of bacteria detected on the surface of H-PE not coated with rGM-CSF. In vitro, rGM-CSF restored the anti-bacterial potency of the phagocytes, which had been reduced by surface contact with H-PE. The results suggest that modification of biomaterials with rGM-CSF could be one way of preventing staphylococcal infections; especially in neutropenic disorders, which constitute the highest risk factor for foreign body-associated infections.     

CNTF overproduction hastens onset of symptoms in motor neuron degeneration (mnd) mice

Ciliary neurotrophic factor (CNTF) promotes the survival of motor neurons, in vitro and in vivo. Moreover, CNTF can block the degeneration of injured or diseased motor neurons in young rodents. Motor neuron degeneration (mnd) mutant mice display adult onset symptoms reflecting progressive motor debilitation and provide a model in which to test the hypothesis that CNTF can prevent the loss of these motor functions. We generated mnd mice that harbor a genomically integrated transgene, resulting in overexpression of the encoded CNTF protein in these mice. In contrast to the beneficial effects of CNTF in preventing motor neuron degeneration in other experimental paradigms, we report that overproduction of CNTF increased the rate of onset of motor disease symptoms in mnd mice and the presence of the transgene correlated with low adult body weight in mnd and wild-type genetic backgrounds. © 1996 John Wiley & Sons, Inc.     

Relative ability of ovine follicle stimulating hormone and itsβ-subunit to generate antibodies having bioneutralization potential in nonhuman primates

The relative ability of ovine follicle stimulating hormone and itsβ-subunit, two potential candidates for male contraceptive vaccine, to generate antibodies in monkeys capable of bioneutralizing follicle stimulating hormone was assessed usingin vitro model systems. Antiserum against native ovine follicle stimulating hormone was found to be highly specific to the intact form with no cross-reactivity with either of the two subunits while the antiserum againstβ-subunit of follicle stimulating hormone could bind to theβ-subunit in its free form as well as when it is combined withα-subunit to form the intact hormone. Both antisera could block the binding of the hormone to the receptor if the hormone was preincubated with the antibody. However, the follicle stimulating hormoneβ-antisera could only inhibit the binding of the hormone partially (33% inhibition) if the antibody and receptor were mixed prior to the addition of the hormone, while antisera to the native follicle stimulating hormone could block the binding completely (100% inhibition) in the same experiment. Similarly antisera to the native follicle stimulating hormone was significantly effective in blocking (100%) response to follicle stimulating hormone but not theβ-subunit antisera (0%) as checked using anin vitro granulosa cell system. Thus the probability of obtaining antibodies of greater bioneutralization potential is much higher if intact hormone is used as an antigen rather than itsβ-subunit as a vaccine. Majority of the work reported here was carried out during the tenure of Visiting Scientist fellowship awarded by the MRC Canada to the first author.     

THE EFFECT OF CYTOSINE ARABINOSIDE IN VITRO ON AGAR COLONY FORMING CELLS AND SPLEEN COLONY FORMING CELLS OF C57BL MOUSE BONE MARROW

This study reports the effect of cytosine arabinoside in culture on two classes of bone marrow progenitor cells in C57BL mice, agar colony forming cells (ACU) and spleen colony forming cells (CFU). Both normal cells and rapidly proliferating cells were studied. The results show that in normal mice, 23 % of ACU but only 7 % of CFU are killed following 1 hr incubation with the drug. With longer periods of incubation, the survival of ACU in the controls is poor, and the results for the drug-treated cultures suggest that the cells are held up in cycle. In continuously irradiated mice, the proportion of ACU and CFU killed after 1 hr incubation with drug is increased to 43–54%, confirming previous results that these cells are proliferating more rapidly than in normal mice. In mice treated with myerlan, 54 % of ACU are killed by 1 hr in vitro exposure to cytosine arabinoside, again confirming that ACU are rapidly proliferating. However, the proportion of CFU killed is lower (23 %). These results are compared with other studies of the effect of cytosine arabinoside in vivo and also with thymidine suicide in the same strain of mice. The results show that cytosine arabinoside has the same effect as tritiated thymidine, and also that the proportion of CFU killed by these agents in vitro is lower than when the agents are injected in vivo. It is suggested that the conditions in culture have an adverse effect on CFU, which cease DNA synthesis, and are protected from the killing effect of cytosine arabinoside and tritiated thymidine. Since cytosine arabinoside in vitro has an effect similar to tritiated thymidine in vitro on bone marrow progenitor cells in C57BL mice, in vitro incubation with cytosine arabinoside could be an alternative method to thymidine suicide for measuring differences in cell proliferation rate.     

PROTECTION OF CYCLING CFUs AGAINST HYDROXYUREA BY LOW DOSES OF ACTINOMYCIN D

Low dose (80 μg/kg) Actinomycin D (AD) produced a significant but transient inhibition of proliferation of the haemopoietic stem cells (CFUs) in chimaeras or in mice regenerating after sublethal irradiation. The same dose of AD had no effect on the resting CFUs population. During the period of proliferation inhibition, CFUs proved to be insensitive to the killing effect of [³H]thymidine in vitro and hydroxyurea (HU) in vivo. In Ehrlich ascites tumour (EAT) bearing mice enhanced CFUs turnover rate was found. Eighty μg/kg AD produced a selective effect in these mice: it protected the proliferating CFUs population without diminishing the effect of hydroxyurea on the tumour cells.